6 beta-hydroxycortisone 6, 21-diacylate compounds and process for the production thereof



United States Patent G B-HYDROXYCORTISONE 6,21-DIACYLATE COM- POUNDS ANDPROCESS FOR THE PRODUC- TION THEREOF Arthur R. Hanze, KalamazooTownship, Kalamazoo County, Herbert C. Murray, Barry Township, BarryCounty, and Oldrich K. Sebek, Kalamazoo, Mich., assignors to The UpjohnCompany, Kalamazoo, Mich., a corporation of Michigan No Drawing.Application November 21, 1958 Serial No. 775,358

5 Claims. (Cl. 260-39745) The present invention relates to a new processfor the production of 6e-hydroxycortisone 6,21-diacylates and to thenovel intermediates, 6,8,21-dihydroxy-4,17(20)-pregnadiene-3,11-dioneand the 6,2l-diacylates thereof.

It is an object of the present invention to provide a process for theproduction of 6fl-hydroxycortisone 6,21- diacylate. Another object is toproduce the novel intermediate, 6,8,21-dihydroxy-4,17(20) pregnadiene3,11- dione and the 6,2l-diacylates thereof. 1 Other objects will beapparent to those skilled in the art to which this invention pertains.

The process of the present invention comprises: aerobically contacting115,21-dihydroxy 4,l7(20) pregnadien-3-one with the oxidizing activityof a species of the fungus Rhizopus in an equeous nutrient medium underagitated, submerged, aerobic fermentation conditions until a substantialamount of 6fl,21-dihydroxy-4,17(20)- pregnadiene-3,11-dioneis produced.The 6,8,21-dihydroxy-4,17(20)-pregnadiene-3,ll-dione is then esterifiedwith an acylating agent, where the acyl radical is of an organic acid,preferably a hydrocarbon carboxylic acid containing from one to twelvecarbon atoms, inclusive, to produce6fi,21-dihydroxy-4,17(20)-pregnadiene-3,11- dione 6,21-diacylate. The6/3,21-dihydroxy-4,17(20)-pregnadiene-3,1l-dione 6,21-diacylate isthereupon treated withosmium tetroxide in the presence of a peroxide,such as hydrogen peroxide or an amine oxide peroxide, such asnnnethylmorpholine oxide peroxide to give 6,8-hydroxycortisone6,21-diacylate.

The GB-hydroxycortisone 6,21-diacylates have antiphlogistic,anti-rheumatoid, anti-arthritic and anti-inflammatory activity. They areuseful in thetreatment of inflammatory conditions of the skin, nose,eyes and ears of humans and domestic animals which are caused, by avariety of bacterial and fungal infections, contact dermathis and otherallergenic reactions. These; compounds also have diuretic activity andsalt losing properties. which make then useful in the alleviation ofedematous conditions resulting from excessive retention of salt andwater. In general, the compounds of the present invention can beprepared for animal or human use by incorporating them in any one ofseveral dosage forms suitable for, such use. Such dosage forms includethe active ingredients plus a non-toxic carrier which can beeither asolid ma terial or a liquid. Bland carriers are preferred for oral use,and examples of oral dosage forms are tablets, capsules, liquidsuspensions or solutions. For the dosage forms which are particularlysuitable for parenteral ad ministration, a sterile diluent is necessary.When the active ingredients are to be used topically they can beincorporated in an ointment, a bougie, a lotion or a jelly. When theintended use is the eye or car, the compounds can be prepared in theform of drops or an ointment. The compounds may also be prepared in anaerosol vehicle when the intended use is nasal.

According to the process of the present invention, the startingmaterial, 1 118,21 -dihydroxy-4, 17 (20) -pregnadien- 3 one, isconverted to 65,21-dihydroxy-4,17(20)-pregna- 2,935,521 Patented, May3,, 1960 diene-3,l1-dione by incorporating the ll-hydroxy startingmaterial in the fermentation medium with the,oxidiz ing activity of aspecies of fungus of the genus Rhizopus. Throughout the genus theproportion of 613,21-dihydroxy- 4,l7(20)-pregnadiene-3,1l-dione productwill vary compared with other oxidized and/or hydroxylated sub stancespresent. Recovery of the above-named product from the fermentationliquors, can be accomplished, by the methods described in the exampleswhich follow. Species in which the proportion of the aboveenamed productis especially high are Rhizopus arrhizus, Rhizopus delema'r,Rhizopus-oryzae, Rhizopus japonicus, Rhizopus shanghniesis,, Rhizopuskazensis, Rhizopus kohnii, Rhizopus tritici, and Rhizopus nigricans.Typical strains. of these organisms, are listed below.

Rhizomes arrhizus A.T.C.C. 11145 (Rh-176) Rhizopus arrhizus NRRL (R-l6)Rhizopus delemar A.T.C.C. 4858 Rhizopus delemar A.T.C.C. 9374 Rhizopusoryzae A.T.C.C. 9363 Rhizopus oryzae A.T.C.C. 10260 Rhizopzls nigricansA.T.C.C. 10404 Rhizopus nigricans A.T.C.C. 7577 Rhi'zopus nigricansA.T.C.C. 6204 Rhizopus japonicus A.T.C.C. 8466 Rhizopus shanghaiesisA.T.C.C. 10329 Rhizopus kazensis A.T.C.C. 8998 Rhizopus kohnii A.T.C.C.8996 Rhizopus tritici A.T.C.C. 1230 The process of the invention iscarried out by contacting the steroid starting material, abovementioned, with the oxidizing activity of a species of fungus of thegenus Rhizopus in an aqueous nutrient medium under agitated, submerged,aerobic fermentation conditions as disclosed in U.S. Patent 2,602,769.This is preferably accomplished by direct fermentation with themicroorganism, but can readily be accomplished by using the oxidizingenzymesthereof as disclosed in the patent.

The 65,21 dihydroxy-4,17 (20) -pregnadiene'-3,1 l-dione isrecovered-from the fermentation reaction mixture and purified byconventional methods, such as for example, extraction with an organicsolvent and recrystallization as disclosed in the cited patent. Thepreferred method is the extraction procedure wtih methylene chloride asdisclosed by D. H. Peterson et al., J. Am. Chem. Soc, 74,- 5933 (1952).The crude solvent-free residue thus obtained can be purified to yield6,8,2l-dihydroxy-4,17 (20)'- pregnadiene-3-,ll-dione by chromatographyor re: crystallization from an organic solvent such as acetone, ethylenechloride, chloroform, ethers, esters, ketones, ethyl acetate, amylacetate and the like.

The esterification step involves the conversion of 65,21-dihydroxy-4,17(20)-pregnadiene-3,11-dione to the corresponding6,21-diacyloxy compound. This reaction can be performed underesterification conditions known in the art, e.g., by the reaction of the6,21-dihydroxy steroid with the selected acid halide, e.g., acidchloride or acid bromide, or the anhydricle of an organic carboxylicacid. Compounds thus produced include the 6,8,21-dihydroxy-4,17(20)-pregnadiene-3,1l-dione 6,21-diacylates wherein the acylradical-is of an organic acid, preferably a hydrocarbon carboxylic acidcontaining from one to twelve carbon atoms, inclusive, e.g., propionic,butyric, isobutyric,

valeric, isovaleric, trimethylacetic, Z-methylbutyric, 3-;

or 4-methylbenzoic, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-dimethylbenzoic, ethylbenzoic, 2,4,6- trimethylbenzoic,

3 2,4,6-triethylbenzoic, u-naphthoic, 3-methy1-a-naphthoic, an aralkylacid, e.g., phenylacetic, phenylpropionic, diphenylacetic,triphenylacetic, an unsaturated acid, e.g., acrylic, maleic, vinylacetic, propiolic, undecolic, etc.

The oxidative hydroxylation of 65,21-dihydroxy-4,17-(20)-pregnadiene-3,1l-dione 6,21-diacylate to produce the corresponding65-hydroxycortisone 6,21-diacylate is carried out by reaction withosmium tetroxide and an oxidizing agent such as hydrogen peroxide,peracids, alkyl peroxides, amine oxide peroxides, and the like. Apreferred procedure involves reaction of 65,21-dihydroxy-4,17(20)-pregnadiene-3,1l-dione 6,21-diacylate with osmium tetroxide andan oxidizing agent, such as an aryl iodoso compound such as phenyliodoso acetate or a tertiary amine oxide peroxide such asN-methylmorpholine oxide peroxide, in an organic solvent. Tertiary butylalcohol is the preferred solvent, but other solvents such as methanol,ethanol, acetone, dioxane, and the like are operative. The reaction canbe conducted either in the light or in the dark and usually at roomtemperature, although temperatures from ten degrees centigrade to theboiling point of the mixture are operative. The time required for thereaction is not critical and can be varied between about one and 72hours, the length of time being dependent on the temperature and theamount of osmium tetroxide and oxidizing agent employed.

The following examples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting.

EXAMPLE 1 65,21 -dihydrxy-4,1 7(20) -pregnadiene-3,I1 -dione A fermentercontaining ten liters of a sterile medium at pH 4.9, consisting of tengrams per liter of commercial dextrose and twenty grams per liter ofcorn steep, was inoculated with 500 milliliters of vegetative growth ofRhizopus arrhizus (A.T.C.C. 11145). After 21 hours of vigorous agitation(300 r.p.m.) and aeration at two liters per minute, a solution of 2.5grams of 115,21- dihydroxy-4,17(20)-pregnadien-3-one in sixtymilliliters of acetone was added, and the fermentation continued for 48hours under the same conditions. The conversion products were thenextracted with methylene chloride and processed in the same manner asdescribed by D. H. Peterson et al., J. Am. Chem. Soc., 74, 5933 (1952),giving about five grams of a crystalline residue. Paper chromatographyusing the FTP system [A. Zaffaroni et al., J. Biol. Chem., 193, 749(1951)], indicated the presence of a new compound with greatly increasedpolarity. The residue was triturated with acetone and filtered, giving610 milligrams of crystals of crude 65,21-dihydroxy-4,l7-(20)-pregnadiene-3,1l-dione. The crude product thus obtained wasdissolved in 65 milliliters of hot acetone, filtered through a bed ofmagnesium silicate filter aid, concentrated to crystallization, filteredand dried, yielding 360 milligrams of65,21-dihydroxy-4,l7(20)-pregnadiene- 3,11-dione melting at 252 to 254degrees centigrade, having an ultraviolet absorption M 233 ru a 16,000and infrared maxima in mineral oil mull of 3500, 3410, 1697, 1667, and1617 reciprocal centimeters.

Analysis.Calculated for C H O (344.43): C, 73.22; H, 8.20. Found: C,73.53; H, 8.47.

The mother liquors from the above crystallizations were combined,concentrated to remove the acetone and chromatographed on 300 grams ofsynthetic magnesium silicate and eluted with ZOO-milliliter fractions asfollows:

TABLE I Fractions Solvents Fractions 17 to 21, inclusive, were combinedand crystallized from acetone to give 500 milligrams of 65,21- dihydroxy4,17(20) pregnadiene 3,11 dione melting at 243 to 247 degreescentigrade, which on recrystallization from acetone gave 400 milligramsof 65,21-dihydroxy- 4,l7(20)-pregnadiene-3,1l-dione melting at 246 to253 degrees centigrade.

Following exactly the procedure described in the paragraphs above, butsubstituting Rhizopus arrhizus NRRL strain (R-16), Rhizopus delemarA.T.C.C. 4858, Rhizous oryzae A.T.C.C. 9363, Rhizopus nigricans A.T.C.C.10404, Rhizopus japonz'cus A.T.C.C. 8466, Rhizopus shanghaiesis A.T.C.C.10329, Rhizopus kazensis A.T.C.C. 8998, Rhizopus kohnii, A.T.C.C. 8996and Rhizopus tritici A.T.C.C. 1230 in successive experiments forRhizopus arrhizus A.T.C.C. 11145 of Example 1, there was obtained65,21-dihydroxy-4,17(20)-pregnadiene-3,1l-dione. The fermentation wascarried out for a duration of 48 hours, the conversion productsextracted with methylene chloride and the crude product recovered asdisclosed above. out also exactly as disclosed above, i.e., bychromatography on synthetic magnesium silicate followed by elution withZOO-milliliter fractions of methylene chloride followed by fractions ofSkellysolve B hexanes containing increasing proportions of acetonewithin the range of about ten to about fifty percent acetone.

EXAMPLE 2 65,21 -dihydroxy-4,1 7 (20) -pregnadiene-3,11-dione6,21-diacetate A solution of 400 milligrams of 65,21-dihydroxy-4,17(20)-pregnadiene-3,1l-dione in one milliliter of pyridine and onemilliliter of acetic anhydride was allowed to react overnight at about25 degrees centigrade. The excess acetic anhydride was then decomposedby the addition of ice water and the resulting crystalline solids wereremoved by filtration and dried, yielding 440 milligrams of crude65,21-dihydroxy-4,17(20)-pregnadiene- 3,11-dione 6,2l-diacetate, meltingat 129 to degrees centigrade. Recrystallization from a solution of onemilliliter of ethyl acetate and two milliliters of Skellysolve B hexanesgave 270 milligrams of 65,21-dihydroxy- 4,17(20)-pregnadiene-3,1l-dione6,21-diacetate having a melting point of 136 to 138.5 degreescentigrade, a specific rotation of [11] plus 66 degrees in dioxane, anultraviolet absorption a 13,250, and infrared maxima in mineral oil mullof 1732, 1700, 1676, 1617, 1230 and 1250 reciprocal centimeters.

Analysis.--Calculated for C H O C, 70.07; H, 7.53. Found: C, 69.70; H,7.90.

Similarly, by allowing 65,21-dihydroxy-4,17(20)-pregnadiene-3,11-dioneto react with the appropriate carboxylic acid anhydride, there areproduced other 65,21- diacylates, such as for example,65,21-dihydroxy-4, 17(20)-pregnadiene-3,1l-dione 6,21-dipropionate,65,21- dihydroxy-4, 17 (20) -pregnadiene-3,1 l-dione 6,21-dibutyrate,65,21 dihydroxy-4,l7(20)-pregnadicne-3,ll-dione 6,21-divalerate,65,2l-dihydroxy 4,17 (20)-pregnadiene- 3,1l-dione 6,2l-dihexanoate,65,21-dihydroxy-4,17(20)- pregnadiene-3,11dione 6,21-dilaurate,65,21-dihydroxy- 4, 17 20) -pregnadiene-3,1 l-dione 6,2l-ditrimethylacetatc. 65,21-dihydroxy-4,17(20) pregnadiene-3,11-dione6,2ldiisobutyrate, 65,21 -dihydroxy-4, 17 (20 -pregnadiene-3, 11--dione6,2 l-diisovalerate, 65,2 1-dihydroxy-4, 17 (20) pregnadiene 3,11-dione6,21 dicyclohexanecarboxylate, 65,21-dihydroxy-4, 17 (20)-pregnadiene-3,1 l-dione 6,21-dibenzoate, 65,21 dihydroxy 4,17(20)-pregnadiene-3,11- dione 6,21-dipheuylacetate,65,21-dihydroxy-4,17(20)- pregnadiene-3,11-dione6,21-di-5-phenylpropionate, 65,21-dihydroxy-4,17(20)-pregnadiene-3,1l-dione 6,21-di-o', mand p-toluate,65,21 dihydroxy 4,17(20) pregnadiene- Purification of the crude productcan be carried 3,11-dione 6,21 dihemisuccinate, 6,9,21 dihydroxy-4,l7(20)-pregnadiene-3,ll-dione 6,2l-dihemiadipate, 6,3,21-dihydroxy-4,17(20)-pregnadiene-3,1l-dione 6,21-diacrylate,66,2l-dihydroxy 4,17(20)-pregnadiene-3,11 dione 6,21-dicrotonate, 65,2I-dihydroxy-4, 17 (20 -pregnadiene- 3,11 dione 6,21 diundecylenate, 63,21 dihydroxy 4,

6,2l-dilaurate, fifihydroxycortisone 6,2l-ditrimethylacetate,fl-hydroxycortisone6,2l-diisobutyrate, 6/3-hydroxycortisone6,21-diisovalerate, 6,8-hydroxycortisone 6,21-di-17(20)-pregnadiene-3,11-dione 6,21-dipropiolate, 6,6,21-

dihydroxy 4,l7(20)-pregnadiene-3,l1 dione 6,21-dicinnamate, 613,21dihydroxy 4,l7(20)-pregnadiene 3,11- dione 6,2l-dimaleate, 6B,21-dihydrxy-4,17(20)pregnadiene-3,l1-dione 6,21-dicitraconate, and the like. 1

, EXAMPLE 3 fifi-hydroxycortisone 6,21'-diacetate 7 To 193 milligrams(0.45 millimole) of 65,21-dihydroxy-4,l7(20)-pregnadiene-3,l1-dione6,2l-diacetate in 7.5 milliliters of tertiary butyl alcohol and 0.2milliliter of pyridine was added 1.13 millimoles of N-methyl-mor pholineoxide peroxide and 1.4 milligrams of osmium tetroxide in 0.75 milliliterof tertiary butyl alcohol. After three hours at room temperature (about25 degrees centigrade) four milliliters of 0.5 percent sodium sulfitewas added and the solution was stirred thirty minutes at roomtemperature. The solution was then concentrated under reduced pressureuntil copious crystallization ocand, equivalents will be apparent to oneskilled in the art,

curred. The mixture was stirred for a period of one hour, filtered andthe solid washed first with 1:4 tertiary butyl alcohol-water, then withWater; the solids were dried, yielding 135 milligrams ofGil-hydroxycortisone 6,2l-diacetate melting at 233 to 237 degreescentigrade. Recrystallization from acetone-Skellysolve B hexanes gave135 milligrams of 6,8-hydroxycortisone 6,21-diacetate having a meltingpoint of 242 to 244.5 degrees centigrade, a specific rotation of [aJbplus 131 degrees in dioxane, an ultraviolet absorption a 13,325, andinfrared maxima in mineral'oil mull of 3540, 1735, 1712, 1697, 1675,1620and 1240 reciprocal centimeters.

EXAMPLE 4 Oxygenation with phenyliodosoacetate To a solution of 1.284grams (3.0 millimoles) of 766,21-dihydroxy-4,17(20)-pregnadiene-3,1l-dione 6,21-di acetatedissolved in sixty milliliters of tertiary butyl a1- cohol and 1.5milliliters of pyridine was added, at degrees centigrade, fivemilliliters of tertiary butyl alcohol containing 11.1 milligrams (0.044millimole) of osmium tetroxide and 0.2 milliliter (0.11 millimole) ofwater. To the solution was then added 2.4 grams (7.5 millimoles) ofphenyliodosoacetate. When the reaction was complete, twenty millilitersof 'water was added to the reaction mixture and then distilled atreduced pressure until copious crystallization occurred. The mixture wasstirred for a period of one hour, filtered and the solid washed, firstwith 1:4 tertiary butyl alcohol-water, then with Water, the solids weredried, yielding 6fl-hydroxycortisone 6,2l-diacetate. Recrystallizationfrom acetone- Skellysolve B hexanes gave substantially pureGB-hydroxycortisone 6,2l-diacetate corresponding to that obtained inExample 3, above.

Similarly, by substituting as starting material other 613,21-dihydroxy-4, 17 (20 -pregnadiene 3,11-di0ne 6,21-

diacylates, prepared in Example 2, the procedures of Examples 3 and 4,above, are productive of other 618-hydroxycortisone 6,21-diacylates,such as for example, 65-- and the invention is therefore to be limitedonly by the scope of the appended claims.

We claim:

l. A process comprising aerobically contacting llfl,2l-dihydroxy-4,l7(20)-pregnadien-3-one with the oxidizing activity of aspecies of fungus of the genus Rhizopus in an aqueous nutrient mediumunder submerged fer mentation conditionsand continuing the fermentationuntil asubstantial amount of 6fi,21-dihydroxy-4,17(20)-pregnadiene-3,11-dione is formed, and recovering the latter compoundfrom the fermentation medium.

2. A process comprising aerobically contacting 11p,2l-dihydroxy.-4,l7(20)-pregnadien-3-one with the oxidizing activity oforganisms of the fungus Rhizopus arrhizus in an aqueous nutrient mediumunder submerged aerobic fermentation conditions and continuing thefermentation until a substantial amount of 6fi,2l-dihydroxy-4,l7(20)-pregnadiene3,l1-dione is formed, and recovering the latter compound fromthe fermentation medium.

7 3. A steroid compound of the formula:

CHs

CHsOR ll 3 OH I 0:

wherein R is the same member selected from the group References Cited inthe file of this patent UNITED STATES PATENTS 2,602,769 Murray et a1.July 8, 1952 2,735,800 Murray et a1 Feb. 21, 1956 2,769,823 Schneider etal Nov. 6, 1956 2,842,568 Herz et al July 8, 1958 2,875,217 SchneiderFeb. 24, 1959 2,899,448 Beal et a1. Aug. 11, 1959 OTHER REFERENCES lbidpages 1331-50 Green CAVol. 50 page 389i UNITED STATES PATENT OFFICECertificate of Correction Patent No. 2,935,521 May 3, 1960 Arthur R.Hanze et a1.

It is hereby certified that error appears in the printed specificationof the above numbered patent requiring correction and that the saidLetters Patent should read as corrected below.

Column 6, lines 39 to 50, the formula should appear as shown belowinstead of as in the patent:

CHgOR or R Signed and sealed this 22nd day of November 1960.

Attesting Ojfiaer. Gonwm'saz'mwr of Patents.

3. A STEROID COMPOUND OF THE FORMULA: